|
| Status |
Public on Apr 22, 2016 |
| Title |
PECAM1plusPDGFRaminExp2 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129X1/SvJ x 129S1/Sv
cell type: undifferentiated ESC culture
passages: 13-17
cell subpopulation: PECAM1plusPDGFRamin
|
| Treatment protocol |
Single-cell suspensions of ESCs were obtained by dissociating with 0cell dissociation buffer (Invitrogen) at 37°C. Cells were incubated with the primary antibodies in PBS for 30 min on ice and sorted using a FACSariaIII
|
| Growth protocol |
ESC were maintained feeder-free on gelatin (EmbryoMax® 0.1% Gelatin Solution, ES-006-B; Millipore) coated plates, in Knockout DMEM (10829–018; Gibco), 20% Knockout Serum Replacement (KSR, 10828-028; Gibco), 2 mM L-glutamine (25030-024; Gibco), 1X minimal essential medium nonessential amino acids (11140-035; Gibco), penicillin (100 U/ml)/streptomycin(100 μg/ml) (15140-122; Gibco), 100 μM β-Mercaptoethanol (31350; Gibco) and 1,000 U/ml recombinant mouse leukemia inhibitory factor (ESGRO, ESG1107; Chemicon International).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from 100000 sorted events for each subpopulation was extracted by using the Mirneasy RNA micro kit (QIAGEN). RNA was amplified by using the Ovation® RNA Amplification System V2 (NUGEN).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina Casava1.8 software used for basecalling.
Low quality ends of sequence reads were trimmed using FastX 0.0.13. Adapters were trimmed with cutadapt 1.2.1. Low quality sequence reads were filtered using FastX 0.0.13 and ShortRead1.20.0. Contaminants were removed using bowtie 2.1.0.
Reads were mapped to the reference genome of Mus musculus (Mus musculus GRCm38.73) using Tophat v2.0.8b with parameters: --library-type fr-firststrand --min-intron-length 50 --max-intron-length 500000 --no-coverage-search --no-mixed --read-realign-edit-dist 3.
The number of reads in the alignments that overlap with the gene features was counted by htseq-count 0.5.4p3 with parameters: -m union --stranded=no -a 0 -t exon -i geneid. Genes for which all samples have less than 1 counts-per-million (absent genes) were filtered.
RPKM (Reads per kilobase of gene sequence and per Million reads of library size) was calculated as following: Counts for each gene were divided by the total number of counts (in million) and by the gene length (in kbp).
Genome_build: Mus musculus GRCm38.73
Supplementary_files_format_and_content: tab-delimited text files of matrix tables of raw counts and RPKM values for all samples
|
| |
|
| Submission date |
Feb 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Antonio Lo Nigro |
| E-mail(s) |
antoniolonigro83@gmail.com
|
| Organization name |
Ismett
|
| Street address |
Via tricomi 5
|
| City |
Palermo |
| ZIP/Postal code |
90134 |
| Country |
Italy |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE65884 |
PDGFRα+ cells in ESC cultures represent the in vitro equivalent of the pre-implantation primitive endoderm precursors |
|
| Relations |
| BioSample |
SAMN03342079 |
| SRA |
SRX876082 |
